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Simple Summary: The Hippo signaling pathway plays a significant role in regulating the organ development processes of mammals. Our research aimed to investigate the expression and distribution of key members of the Hippo signaling pathway in yak mammary glands during different stages. Using immunohistochemistry, Western blot, and relative quantitative real-time polymerase chain reaction techniques, we found that the protein and mRNA expression levels of MST1, LATS1, YAP1 and TEAD1 in the yak's mammary gland varies with the growth, lactation, and dry periods. The differential expression in the yak's mammary gland at different stages strongly suggests that the Hippo signaling pathway plays an important role in regulating the mammary gland development processes under different physiological conditions. Due to its rich nutritional value, yak milk is an important food source in the alpine pastoral areas. However, yaks have a low milk yield. The Hippo pathway participates in cell proliferation and organ development. We aimed to determine the regulatory mechanism of this pathway in yak mammary cells. A greater understanding of how the expression of its essential genes influence the reproductive cycle could lead to improvements in lactation performance. The expression levels of the key genes MST1, LATS1, YAP1, and TEAD1 were detected by quantitative real-time PCR, Western blotting, and immunohistochemistry during the growth, lactation, and dry periods (GP, LP and DP, respectively). The MST1 and LATS1 mRNA and protein expression level was highest during GP and lowest during LP. The YAP1 and TEAD1 mRNA and protein expression level decreased from GP to LP and DP. MST1 and LATS1 were expressed in the cytoplasm whereas YAP1 and TEAD1 were expressed in the nucleus and cytoplasm, respectively. The differential expression of MST1, LATS1, YAP1, and TEAD1 expression in the yak mammary gland during different developmental stages strongly suggests that they play an important role in the regulation of developmental functions under different physiological conditions. [ABSTRACT FROM AUTHOR]

The ultimate fate of Graafian follicles is ovulation or atresia which relies on the highly coordinated processes of apoptosis and autophagy in ovarian cells. Long non-coding RNA maternally expressed gene 3 (LncRNA MEG3), miR-23a, and apoptosis signal-regulating kinase 1 (ASK1) are factors associated with autophagy. However, whether these factors can regulate autophagy in cumulus cells (CCs) of yak is unclear. Here, miR-23a overexpression upregulated the LC3-II/LC3-I ratio and Beclin1 abundance while reducing p62 accumulation (p < 0.05). The monodansylcadaverine assay exhibited a marked increase in punctate green fluorescence, and the GFP-LC3B displayed increased yellow fluorescence (p < 0.05). The opposite effect was observed for miR-23a inhibitors. Furthermore, miR-23a overexpression downregulated the abundance of ASK1 mRNA and total ASK1 protein (t-ASK1), whereas miR-23a inhibitors up-regulated them (p < 0.05). The effects of miR-23a overexpression on ASK1 phosphorylated protein at serine 845 (P-845), total JNK (c-Jun N-terminal kinase) (t-JNK) and the JNK phosphorylated protein (p-JNK) were similar to those of t-ASK1 but elicited the opposite effect on ASK1 phosphorylated protein at serine 967 (P-967) (p < 0.05). We further demonstrated that ASK1 expression can be silenced by small-interfering RNA (siRNA), which had no significant effect on t-JNK abundance (p > 0.05) but significantly suppressed the p-JNK expression (p < 0.05). Silencing ASK1 significantly improved Beclin1 abundance and the LC3-II/LC3-I ratio, but decreased p62 abundance (p < 0.05). An increase in yellow GFP-LC3B puncta and green MDC staining puncta were observed (p < 0.05). Overexpression of LncRNA MEG3 significantly increased the expression of t-ASK1, P-845, and JNK and decreased the abundance of P-967 and miR-23a (p < 0.05). In addition, miR-23a upregulation reduced the number of the TUNEL-positive cells, and the addition of 8 mM 3-methyladenine (3-MA) reversed this downregulation (p < 0.05). Similar trends were observed for the Bax/Bcl2 ratio and cleaved-caspase3 abundance. In summary, miR-23a promotes autophagy by inhibiting ASK1 abundance, which reduces apoptosis of yak CCs. This effect can be inhibited by LncRNA MEG3, which has implications for decreasing abnormal Graafian follicular atresia and maintaining development. • LncRNA MEG3, miR-23a, and ASK1 are closely related to follicular atresia, yet their effect on yak Graafian follicular is unknown. • Overexpression of miR-23a enhances the levels of autophagy in yak cumulus cells (CCs) via downregulation of ASK1/JNK axis. • LncRNA MEG3 overexpression significantly attenuated the downregulation effect of miR-23a on ASK1/JNK axis. • Overexpression of miR-23a inhibits apoptosis, which is caused by miR-23a-induced autophagy. • Effects of LncRNA MEG3, miR-23a, and ASK1 on yak CCs have implications for decreasing abnormal Graafian follicular atresia. [ABSTRACT FROM AUTHOR]

Colony-stimulating factor 2 (CSF 2 ) is known to promote the development and survival of rodents and ruminants preimplantation embryos; however, the effect of CSF 2 on yak embryos has not been reported. The objective of this study was to investigate the effects of CSF 2 on the developmental competence of yak embryos cultured in vitro in modified synthetic oviduct fluid (mSOF) medium and on the expression pattern of heat shock protein 70 kDa 1A (HSPA1A). In each experiment, cumulus-oocyte complexes (COCs) were matured in vitro and fertilized with frozen-thawed semen. Zygotes were treated with varying concentrations of CSF 2 (0, 10, 50, 100 ng/mL) until day 8 after fertilization. Embryo development was calculated as the percentage of oocytes that formed embryos at the 2-cell, 4-cell, 8-cell, 16-cell, morula and blastocyst stages. The total cell numbers (TCN) per blastocyst and their allocation to the inner cell mass (ICM) and trophectoderm (TE) lineages were determined using differential CDX2 staining. The expression of HSPA1A was examined by quantitative real-time PCR (qRT-PCR) and immunochemistry to determine the mRNA and protein levels. The results showed that treatment with 50 ng/mL CSF 2 significantly ( P < 0.05) increased the rate of blastocyst formation (19.01% versus 9.93%) and the TCN per blastocyst (96.94 versus 81.41) compared to the control group. However, no significant differences were observed in the other stages of development. qRT-PCR analysis confirmed that treatment with 50 ng/mL CSF 2 significantly ( P < 0.05) inhibited the expression of HSPA1A mRNA in blastocysts cultured in vitro relative to the control group, but there were no significant differences between the other treatment groups. Immunocytochemical analysis confirmed that HSPA1A protein accumulation was gradually reduced in yak blastocysts cultured in 0, 10, 100 or 50 ng/mL CSF 2 , however, no significant differences were observed between the 10 and 100 ng/mL treatments ( P > 0.05). In conclusion, these findings demonstrate that CSF 2 inhibits the expression of HSPA1A to facilitate yak blastocyst formation and increase cell numbers. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate whether developmental competence of mature vitrified–warmed yak ( Bos grunniens ) oocytes can be enhanced by supplemented insulin-like growth factor I (IGF-1) during in vitro maturation (IVM), and its relationship with the expression of cold-inducible RNA-binding protein (CIRP). In experiment 1, immature yak oocytes were divided into four groups, and IVM supplemented with 0, 50, 100 and 200 ng/mL IGF-1 was evaluated; the mRNA and protein expression levels of CIRP in mature oocytes in the four groups were evaluated using quantitative real-time PCR and western blotting analyses. In experiment 2, the mature yak oocytes in the four groups were cryopreserved using the Cryotop (CT) method, followed by chemical activation and in vitro culture for two days and eight days to determine cleavage, blastocyst rates, and total cell number in the blastocysts. Mature yak oocytes without vitrification served as a control group. The outcomes were as following: (1) the expression of CIRP in the matured oocytes was up-regulated in the IGF-1 groups and was highest expression was observed in the 100 ng/mL IGF-1 treatment group. (2) In the vitrified–warmed groups, the rates of cleavage and blastocyst were also highest in the 100 ng/mL IGF-1 treatment group (81.04 ± 1.06%% and 32.16 ± 1.01%), which were close to the rates observed in groups without vitrification (83.25 ± 0.85% and 32.54 ± 0.34%). The rates of cleavage and blastocyst in the other vitrified–warmed groups were 70.92 ± 1.32% and 27.33 ± 1.31% (0 ng/mL); 72.73 ± 0.74% and 29.41 ± 0.84% (50 ng/mL); 72.43 ± 0.61% and 27.61 ± 0.59% (200 ng/mL), respectively. There was no significant difference in the total cell number per blastocysts between the vitrified–warmed groups and group without vitrification. Thus, we conclude that the enhancement in developmental competence of mature yak vitrified–warmed oocytes after the addition of IGF-1 during IVM might result from the regulation of CIRP expression in mature yak oocytes prior to vitrification. [ABSTRACT FROM AUTHOR]